Ted complicated sample (0.26) was also subjected to measurement for comparison of the absorption peak profiles.More file 3: Preparation of rFab’-MTZ. a) SDS-PAGE evaluation of pepsin digestion of whole rabbit IgG. Lanes: M, molecular-weight size markers; 1, before digestion; 2, just after digestion. b) Fractionation by highperformance size-exclusion chromatography. Panels: left, rF(ab’)2, peak fraction shown in the underbar was collected; appropriate, rFab’-MTZ, peak fraction shown in the underbar was collected. Retention time of each and every peak is shown. (PPTX 231 kb) Abbreviations hFasLECD: human Fas ligand extracellular domain; hFasRECD: human Fas receptor extracellular domain; hFasRECD-Fc: a fusion protein composed of human Fas receptor extracellular domain and human IgG1-Fc domain; MTZ: 6-methyl-1, 2, four, 5-tetrazine group; NaCl: sodium chloride; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel-electrophoresis; TCO: trans-cyclooctene group; Tris-HCl: tris(hydroxymethyl)aminomethane hydrochloride Acknowledgements The authors thank the persons in charge of inquiries about the industrial solutions employed within this study for supplying detailed info on them. Funding This work was supported by a grant for operating expenses in the Ministry of Economy, Trade and Business, Japan. Availability of information and supplies The authors declare that all relevant data are integrated inside the short article and its more files. Authors’ contributions MM created the study, performed experiments, and wrote the manuscript.SARS-CoV-2 3CLpro/3C-like protease Protein Formulation MM and KH analyzed and interpreted the experimental information. All authors read and approved the final manuscript.Envelope glycoprotein gp120 Protein Biological Activity Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests.Added filesAdditional file 1: Preparation of NFK3G1CG4-hFasLECD. a) Gene structure of expression unit and detailed tag sequences. In the tag sequences, the introduced 3 lysine residues and the reactive cysteine residue utilised for chemical modification with either TCOPEG3-MAL or MTZ-PEG4-MAL are shown in blue and red, respectively. AOX-1 P, P. pastoris alcohol oxidase 1 promoter region; -Prepro, Saccharomyces cerevisiae -factor secretion-signal sequence; Tag, tag sequence; hFasLECD (13981, N184Q, N250Q), human Fas ligand extracellular domain containing deletion mutation from residue 103 to 138 and double substitution mutation (N184Q and N250Q); AOX-1 TT, P. pastoris alcohol oxidase 1 transcription termination region. b) Three dimensional structure of hFasLECD-hDcR3 complicated [26]. A biological unit image composed of a single hFasLECD trimer (yellow) plus a triply bound hDcR3 monomer (white) is depicted as space filling models. The N-terminal residues of hFasLECD subunits within this model are shown in green.PMID:34337881 Left panel, a horizontal view. One of several position of N-terminal tag sequence attachment web-sites is arrowed. Appropriate panel, a vertical view. The structure was drawn applying the atomic coordinates (ID: 4smv) and also the graphic software (jV) supplied by Protein Data Bank Japan (PDBj). c) SDS-PAGE analysis of initial stepwise salt-gradient fractionation with the materials in P. pastoris culture medium employing a cation-exchange column (Hi-Trap S 5 ml). Basal buffer: 50 mM sodium acetate (pH 5.5). Lanes: M, Molecular-weight size markers; 1, just before fractionation; two, flow-through fraction; three, 0 mM NaCl fraction; four, 50 mM NaCl fraction; five, 300 mM NaCl fraction; six, 500 mM NaCl fraction. AO.